User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base

From OpenWetWare

Jump to: navigation, search
Project name Main project page

11/18/09

  • Unfinished line item
  • ✓ Finished line item

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH81 (X/P) 0.519 1.67 25.9
2. huPcCD (E/S) 1.590 1.76 79.5
3. mPcCD (E/S) 1.604 1.76 79.5
4. flyPcCD (E/S) 0.903 1.66 45.2
5. fshPcCD (E/S) 1.482 1.74 74.1
6. hCBX8 (E/S) 0.691 1.74 34.5
7. flyPc (E/S) 0.118 1.44 5.9
2. KAH74 (E/X) 0.188 1.37 9.4


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations (*Note: use insert + old backbone in calculations)

Ligation Plate results (lig : neg crtl) 11/19/09
1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng KAH86 >10:1 (Pick 2)
2. KAH48(S/P)/ 20 ng  
3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng KAH87 >10:1 (Pick 2)
4. mPcCD(E/S)/3386*, > 42 ng + " KAH88 >10:1 (Pick 2)
5. flyPcCD(E/S)/3386*, > 42 ng + " KAH89 >10:1 (Pick 2)
6. fshPcCD(E/S)/3386*, > 42 ng + " KAH90 >10:1 (Pick 2)
7. hCBX8(E/S)/1170, > 42 ng + " KAH91 >10:1 (Pick 2)
8. hCBX8(E/S)/1170, > 42 ng + " KAH92 >10:1 (Pick 2)
9. KAH74(S/P)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.4 --- 1.7 1.7 1.7 1.7 0.5 2.5 ---
Vector DNA 0.6 0.6 2.5 2.5 2.5 2.5 2.5 2.5 ---
2x lgn buf (Roche) 5.0 5.0 5.5 5.5 5.5 5.5 5.5 5.5 5.5
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 2.0 3.4 0.3 0.3 0.3 0.3 1.5 --- 2.0
  10 μL 10 μL 11 μL 11 μL 11 μL 11 μL 11 μL 11.5 μL 11 μL

Oligo annealing

  1. NLS myc stop (KAH011)
  2. K-stop (KAH012)
  3. Adapter BglII/EcoRI
  4. Adapter PstI/BglII


DNA (oligos, 100 μM) up to 18 μL
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



Personal tools